AlphaLISA SureFire Ultra Human Total INSM1 Detection Kit, 10,000 Assay Points

Total-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.

Specific expression of INSM1 in NCI-H69 cells

Adherent cells were grown to confluence in a T175 flask at 37°C, 5% CO₂, and were lysed with Lysis Buffer at a density of 500,000 cells/mL. Suspension cells were washed and lysed with 5X Lysis Buffer at a density of 200,000 cells/mL.

INSM1 levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 5,000 adherent cells and 2,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Total INSM1 protein was only detected in NCI-H69 lung cancer cells. Negative expression was observed in all cell lines tested in agreement with the literature.

Sensitivity of Total INSM1 assay

Cell lysate was prepared from NCI-H69 cells cultured for four days in a T175 flasks at 37°C, 5% CO₂. Cells were harvested, washed with HBSS + 0.1% BSA and lysed at 0.5 x 10⁶ cells/mL with the addition of 5X Lysis Buffer.

Lysate was serially diluted in Lysis Buffer and INSM1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. This assay can detect INSM1 expression in less than 50 cells/datapoint.